The endothelial cell is the interfacing cell between the blood vessel lumen and the blood. It has numerous functions including selective vessel permeability; synthesis of prostaglandins, Factor VIII antigen, and fibronectin; metabolism of chylomicrons; and the processing of several vasoactive materials such as bradykinin, serotonin and norepinephrine. The endothelial cell also synthesizes an "endothelium-derived relaxing factor" that is essential for maintenance of normal vascular homeostasis. This endothelium-derived relaxing factor has been identified by Palmer et al., Nature, 327:524-526 (1987), as nitric oxide. Myers et al. Nature 345:161-163 (1990), have further identified the endothelium-derived relaxing factor as S-nitrocysteine, a compound closely related to nitric oxide.
Nitric oxide is synthesized from the guanidino nitrogens of L-arginine, as described by Palmer et al. Nature 333:664-666 (1988). Nitric oxide synthase is the enzyme responsible for the production of nitric oxide from arginine. Nitric oxide synthase has recently been identified in brain by Bredt and Snyder, Proc. Natl. Acad. Sci. 87:682-685 (1990); in macrophages by Hibbs et al., Science 235:473 (1987); in neutrophils by Yui et al., J. Biol. Chem. 266:3369-3371 (1991); in hepatocytes by Knowles et al. Biochem. J. 270:833-836 (1990); in vascular smooth muscle by Wood et al. Biochem. Biophys. Res. Commun. 170:80-88 (1990), and in other tissues. Nitric oxide synthesis requires tetrahydrobiopterin, flavin adenine nucleotide, and NADPH as co-factors. The brain and endothelial cell nitric oxide synthase enzymes require calcium and calmodulin while the regulatory factors required by nitric oxide synthase in the macrophage and neutrophil are less well defined. The brain and macrophage nitric oxide synthases are cytosolic in location. However, endothelial cell nitric oxide synthase is membrane bound, as described by Forstermann, et al., Proc. Natl. Acad. Sci. 88:1788-1792 (1991). The numerous differences between brain, macrophage, and endothelial cell nitric oxide synthase indicate that the molecular structures of these isoforms are different. Recently, Bredt et al., Nature 351:714-718 (1991), cloned and sequenced cDNA encoding brain nitric oxide synthase, and both Xie et al., Science 256:225-228 (1992), and Lyons et al., J. Biol. Chem. 267:6370-6374 (1992), cloned and sequenced macrophage nitric oxide synthase cDNA. Attempts to use the sequence encoding brain nitric oxide synthase as a probe for expression of the enzyme in endothelial cells have been completely unsuccessful, confirming that the nitric oxide synthase expressed by these different cells are structurally distinct.
It is therefore an object of the present invention to provide nucleic acid and amino acid sequences encoding an endothelial cell nitric oxide synthase.
It is a further object of the present invention to provide a probe for nitric oxide synthase mRNA as a diagnostic tool and for studying the function and disorders involving this enzyme.
It is a further object of the present invention to transfect the gene for endothelial cell nitric oxide synthase in vivo to increase local production of nitric oxide in a blood vessel.